Epigenetic regulation of programmed genome instability in O. trifallax

描述（由申请人提供）：DNA的胞嘧啶甲基化是一种抑制性染色质标记，对于哺乳动物发育期间的基因沉默很重要，癌细胞经常通过异常甲基化其启动子来沉默肿瘤抑制基因。当DNA甲基化模式在癌症或发育性疾病中被破坏时，基因组稳定性受到损害。尽管DNA甲基化在基因表达和基因组稳定性中起着关键作用，但指导从头DNA甲基化的机制仍然不清楚。尖毛藻是一种单细胞真核生物，在发育过程中会发生剧烈的基因组重排，将其微核（MIC）基因组转化为分化的大核（MAC）。这个过程是通过精确消除95%的基因组来完成的，并概括了干细胞的关键特性：自我更新（始终保留一个MIC拷贝）和分化（MAC形成）。有趣的是，我在Landweber实验室的工作表明，这种生物在消除过程中使用了从头DNA甲基化。大多数尖毛藻基因组被甲基化并被消除的事实使其成为了解甲基化如何靶向的有吸引力的模型系统：异常甲基化和随后消除5%保留的DNA将是致命的。此外，这种纤毛虫基因组重排的可重复性使其成为研究DNA甲基化和基因组稳定性之间联系的独特模型，这两者通常与癌症和发育过程有关。目标1：通过显微注射人工构建的体外甲基化染色体到营养细胞的MAC中，检验甲基化诱导尖毛藻DNA降解的假设。目标2：甲基-DNA免疫沉淀和高通量测序（meDIP-Seq）将用于确定在尖毛藻基因组重排期间从头DNA甲基化的内源性结构域。目标3：将通过使用甲基转移酶的化学抑制剂分析尖孢属基因组重排中胞嘧啶甲基化的功能相关性，然后对来自处理细胞的DNA进行深度测序。目的4：利用甲基胞嘧啶染色质IP（me-ChIP）和质谱分析技术鉴定新的甲基化途径蛋白。Oxytricha trifallax提供了一个前所未有的机会，研究DNA甲基化在模型系统中的作用，该模型系统已经进化出一个精心设计的基因组重排过程。目前，对人类疾病中从头甲基化的靶向作用知之甚少，因此来自Oxytricha的见解将与人类健康和疾病的研究直接相关。在纤毛虫模型中鉴定的任何保守的候选蛋白或基序可以在人类组织培养或小鼠癌症模型中测试潜在的致癌作用。

DESCRIPTION (provided by applicant): Cytosine methylation of DNA is a repressive chromatin mark important for silencing genes during mammalian development, and cancer cells frequently silence tumor suppressor genes by aberrantly methylating their promoters. Genome stability is compromised when DNA methylation patterns are disrupted in cancer or in developmental disease. Despite the critical role of DNA methylation in gene expression and genome stability, the mechanisms guiding de novo DNA methylation remain poorly defined. Oxytricha trifallax is a unicellular eukaryote that performs dramatic genome rearrangements in a developmental process that transforms its micronucleus (MIC) genome into a differentiated macronucleus (MAC). This process is accomplished by the precise elimination of 95% of the genome, and recapitulates the key property of stem cells: self-renewal (one copy of the MIC is retained at all times) and differentiation (MAC formation). Interestingly, my work in the Landweber lab has shown that this organism uses de novo DNA methylation in the elimination process. The fact that most of the Oxytricha genome is methylated and eliminated makes it an attractive model system to understand how methylation is targeted: aberrant methylation and subsequent elimination of the 5% retained DNA would be fatal. In addition, the reproducibility of genome rearrangements in this ciliate makes it a unique model for studying the connection between DNA methylation and genome stability, both of which are relevant to cancer and developmental processes generally. AIM 1: Test the hypothesis that methylation induces DNA degradation in Oxytricha through microinjection of artificially constructed, in vitro methylated chromosomes into the MAC of vegetative cells. AIM 2: Methyl-DNA immunoprecipitation and high throughput sequencing (meDIP-Seq) will be used to define the endogenous domains of de novo DNA methylation during genome rearrangements in Oxytricha. AIM 3: The functional relevance of cytosine methylation in Oxytricha genome rearrangements will be analyzed by use of chemical inhibitors of methyltransferases, followed by deep sequencing the DNA from treated cells. AIM 4: Identification of novel methylation pathway proteins by methyl-cytosine chromatin IP (me-ChIP) and mass-spec analysis. Oxytricha trifallax provides an unprecedented opportunity to study the role of DNA methylation in a model system that has evolved an elaborate genome rearrangement process. Currently, the targeting of de novo methylation in human disease is poorly understood, so insights from Oxytricha will be immediately relevant to studies of human health and disease. Any conserved candidate proteins or motifs identified in the ciliate model can be tested in human tissue culture or mouse cancer models for potential oncogenic roles.